Extended Data Fig. 2: Genetic validation of Pen1.
From: PEN1 catalyses RNA primer removal during plastid DNA replication in maize
a, Mapping-by-sequencing of pen1 (Top: SNP-index of the mutant-segregated pool; middle: SNP-index of the WT-segregated pool; bottom: the difference in SNP-index values between the two pools). The purple lines indicate the 99% confidence interval, and the orange lines indicate the 95% confidence interval. The red line indicates the ΔSNP-index values. b,c, RT-qPCR analysis of Pen1 expression pattern during various stages of seed development and the maternal tissue. Data are presented as individual values and means ± SD (n = 3, biological replicates). d, Construction of CRISPR/Cas9-mediated Pen1 knockout lines in the C01 inbred line genetic background. Two sgRNAs that specifically target Pen1 were designed, resulting in the identification of three allelic mutants: pen1CR-1, pen1CR-2, and pen1CR-3. e, Immunoblotting analysis of PEN1 in 14-DAP endosperms of C01, pen1cr-1, pen1cr-2 and pen1cr-3. ACTIN was used as an internal control. Three replicates, consistent results. f, Plant phenotypes of the segregated homozygous mutants from self-pollinated pen1CR-1/+, pen1CR-2/+, and pen1CR-3/+ ears. Scale bars: 25 cm. g, Ear phenotypes of the self-pollinated pen1CR-1/+, pen1CR-2/+, and pen1CR-3/+. The white arrows indicate the segregated homozygous mutant kernels. Scale bars: 1 cm. h, The allelic test of pen1 and other alleles. From panels 1 to 3, the pen1/+ ears were pollinated by pen1CR-1/+, pen1CR-2/+, and pen1CR-3/+, respectively. The white arrows indicate the segregated homozygous mutant seeds. From panels 4 to 6, the pen1F3 ears were pollinated by pen1CR-1/+, pen1CR-2/+, and pen1CR-3/+, respectively. Scale bars: 1 cm.
